The study population consisted of patients that were treated by Endodontics Department of the University Hospital of Florence Careggi, Italy. Subject inclusion criteria were: patients aged between 20 and 60 years, able and willing to sign a consent form, single-rooted teeth with necrotic pulp confirmed by electric vitality test, associated with healthy periodontium, physiologic sulcus depth (<3 mm), and absence of bleeding on probing of the involved teeth.